Simultaneous expression of multiple genes in mammalian cells at finely controlled amounts or ratios is required for applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells [Trowitzsch, S. et al. (2011) Bioessays, 33, 946-955; Bieniossek, C. et al. (2012) Trends in Biochemical Sciences, 37, 49-57]. Three common strategies for controlling multiple gene expression in mammalian cells are (i) co-transfection of multiple vectors at different relative amounts [Schlatter, S. et al. (2005) Biotechnology Progress, 21, 122-133], (ii) single vector having promoters with different strength [Yahata, K. et al. (2005) J. Biotechnol., 118, 123-134] or applying different polyadenylation signals to each gene [Yang, Y. S. et al. (2009) Biotechnology And Bioengineering, 102, 1152-1160], and (iii) insertion of splicing signals with varied splicing efficiencies between genes [Fallot, S. et al. (2009) Nucleic Acids Research, 37]. Co-transfection is an inaccurate approach as the relative amount of different genes incorporated into cells varies from cell-to-cell due to variations in transfection efficiency [Chusainow, J. et al. (2009) Biotechnol Bioeng, 102, 1182-1196; Ho, S. C. L. et al. (2012) Journal of Biotechnology, 157, 130-139]. Using a single vector with multiple promoters ensures introduction of different genes into each cell at identical amounts and provides accurate control of gene expression in transient transfections [Yahata, K. et al. (2005) J. Biotechnol., 118, 123-134]. However, the expression ratio between the products of the different genes still varies between cells in a stably transfected cell pool [Lee, C. J. et al. (2009) Biotechnology And Bioengineering, 102, 1107-1118] as the arrangement of multiple promoters in close proximity causes transcriptional interference, where the active expression of one gene suppresses expression of the other genes. Moreover, the degree of suppression of each gene depends on the integration site in the genome [Eszterhas, S. K. et al. (2002) Molecular and Cellular Biology, 22, 469-479]. The use of splicing signals allows stricter control of relative gene expression in both transient and stable transfections as all genes are expressed in one transcript [Fallot, S. et al. (2009) Nucleic Acids Research, 37]. Nonetheless, this method is difficult to use because cryptic splicing sites in protein coding sequences need to be eliminated.
Co-expression of multiple genes from one mRNA for strict control of the relative gene expression can also be achieved by using either 2A elements or internal ribosome entry site (IRES). 2A linked genes are expressed in one single open reading frame (ORF) and “self-cleavage” occurs co-translationally to give equal amounts of co-expressed proteins [de Felipe, P. et al. (2006) Trends in Biotechnology, 24, 68-75]. This method does not allow modulation of the expression ratio between the proteins of interest. Moreover, incomplete cleavage of 2A peptides often results in the attachment of unwanted residues to the proteins of interest and formation of fusion proteins [Ho, S. C. L. et al. (2013) Plos One, 8, e63247].
When IRES elements are included between multiple ORFs, the first ORF is translated by the canonical cap-dependent mechanism while the rest are translated through a cap-independent mechanism [Chan, H. Y. et al. (2011) PLoS One, 6]. Encephalomyocarditis virus (EMCV) IRES is the most widely used IRES for multiple gene expression in mammalian cells because of its superior activity in different cell lines and ability to mediate accurate translation [Bochkov, Y. A. and Palmenberg, A. C. (2006) Biotechniques, 41, 283-284, 286, 288 passim.]. The region that contributes to efficient EMCV IRES translation contains twelve AUGs triplets [Duke, G. M. et al. (1992) Journal of Virology, 66, 1602-1609]. Translation initiation occurs primarily at the 11th AUG (AUG-11), partially at the 12th AUG (AUG-12), and almost none at the 10th AUG (AUG-10) [Kaminski, A. et al. (1994) Embo Journal, 13, 1673-1681].
It has been shown that IRES allows strict control of the relative gene expression in both transient and stable transfections [Ho, S. C. L. et al. (2012) Journal of Biotechnology, 157, 130-139]. In contrast to the 2A element, products generated using IRES does not form any undesirable fusion proteins [Ho, S. C. L. et al. (2013) Plos One, 8, e63247]. More importantly, as genes are translated independently, the relative expression of different genes can be adjusted by varying the strength of IRES applied on each gene. Using naturally available IRES could be a choice, but the modulation range of the expression levels for the different genes is narrow due to the lack of sufficient IRES elements [Sasaki, Y. et al. (2008) Journal of Biotechnology, 136, 103-112]. Generation of a synthetic IRES library based on random mutagenesis can widen the range of IRES activity. In the prior art a set of eleven IRES mutants was generated by error prone PCR which allows controlled gene expression level across a 20-fold range [Livak, K. J. and Schmittgen, T. D. (2001) Methods, 25, 402-408]. However, the strengths of these IRES mutants appear to be cell specific as the relative expression of four IRES mutants significantly varied between expression in HEK293T cells and CHO K1 cells.
Thus, no multiple gene expression controlling system that is based on IRES elements is known in the art wherein the different genes can be controlled individually over a wide range of relative expression and which demonstrates stable expression in different cell lines. Nonetheless, there is need in the art for such system in synthetic biology and cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells.